The aim of this research program is to investigate the anti- hemostatic and anti-inflammatory compounds in the saliva of blood-feeding insects and ticks which allow efficient blood feeding and enhancement of pathogen transmission. Anti-hemostatic compounds of interest include anti-clotting, anti- platelet and vasodilators. Anti-inflammatory compounds include immunomodulatory compounds as well as compounds that modify effector arms of the immune response, such as anti-complement activity found in the saliva of some ticks. While the vector attempts to modify the feeding site to enhance success of blood feeding, such site becomes locally compromised in its ability to react to injury and becomes an easy site for pathogen invasion. On the other hand, when the vertebrate mounts an immune response to these salivary compounds, pathogen transmission may be compromised. Novel pharmaceuticals and novel targets for vaccine development will be ultimate benefits of this program. The work involves obtaining saliva or salivary glands of the insects and ticks under study, bioassay of their biological activity using both in vivo and in vitro tests, purification of the individual activities using chromatographic and electrophoretic techniques, microsequencing of the peptides and proteins, and final identification of the total primary sequence by molecular biology methods. Confirmation of the clones is found by expression in bacterial or eukaryotic cell lines. We have initiated a reverse approach where salivary cDNA libraries from blood-feeding insects and ticks are being mass sequenced, providing new insights in the discovery of novel compounds. In the current fiscal year we continued on the discovery of gene products that are expressed in the salivary glands of important vectors of human disease, by extending the sialo transcriptome coverage of the mosquitoes Anopheles gambiae and describing for the firt time the sialo transcriptome of the tick vector of Lyme disease in the West coast, Ixodes pacificus. We also described the transcriptome of the venomous organs of a poisonous caterpillar. With this approach, we discovered novel salivary enzymatic activities, novel properties of known salivary proteins, and novel anti-coagulants. In particular, we described poteint anti-angiogenic activity in tick saliva, a potent anti-inflammatory activity in sand fly saliva in collaboration with With Dr. Fernando Cunha (State University of Sao Paulo), characterized the mode of action of one tick anticlotting molecule, and determined the structural determinants of the anticlotting function in one triatomine salivary protein. Our bioinformatic capability also allowed us to maintain and update a database on the proteome of the mosquito Anopheles gambiae (AnoXcel), and to collaborate extensively with other research groups inside and outside the NIAID. We finalized the bioinformatic analysis of mosquito EST and microarray data from the laboratories of Dr. Marcelo Jacobs-Lorena, George Dimopoulos from the Johns Hopkins University, and Drs. Osvaldo Marinotti and Antony James from U.C Irvine. We helped in genome analysis of Plasmodium falciparum and Leishmania brasiliensis with Drs. X. Su and A. Cruz, from NIAID and Ribeirao Preto, Brazil. We also finalyzed the analysis of transcriptomes of the liver form of P. yoelli with Dr. John Sacci, University of Maryland. W We are also collaborating with our expertise to Dr. Steve Hoffman's aim of producing an attenuated sporozoite vaccine to protect humans from malaria, in particular to determine salivary gland contaminants in such preparations.